Skip to main content
Fig. 1 | Plant Methods

Fig. 1

From: Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton

Fig. 1

Strategies of constructing the pooled sgRNAs assemblies for cotton genetic transformation. a Amplifying the 29 oligo sgRNAs by PCR and PCR products obtained double-stranded sgRNAs. b Circular vector and linearized vector. The vector was linearized by BsaI restriction enzyme, the removed part is 300 bp in size. The cut run faster than the control sample (CK) then the linearized sample was purified and used for ligation step. c In-fusion reaction contains double-stranded sgRNAs and linearized plasmid. d The recombined vector harboring the double-stranded sgRNAs. e Clones resulted from the transformation to E.coli. f Extraction of plasmid from Kanamycin-resistant clones. The size of the resulted positive clones is less than 100 bp. g Clones resulted from the transformation to Agrobacterium. h High throughput sequencing for the pooled assembly of the constructed vectors harboring the targeted sgRNAs. i Large scale sequences in Agrobacterium and coverage of sgRNAs. Each star indicates reads of every sgRNA included in this study (This figure was created by BioRender web tool)

Back to article page

Plant Methods

ISSN: 1746-4811